IntroductionIn guiding human embryonic stem cell (hESC) technology toward the clinic, 1 key issue to be addressed is a lack of standardization in the culture and maintenance of hESCs. In the absence of mouse embryonic fibroblast (MEF) feeder layers, many researchers rely on "conditioning" in which medium is first exposed to MEFs to acquire soluble factors that support the propagation of undifferentiated hESCs in culture. It has been difficult to discern how MEF conditioning enables hESCs to maintain an undifferentiated state. Other common features of more recently developed hESC culture conditions include the presence of fibroblast growth factor-2 (FGF2), the absence of serum, and the presence of a serum substitute such as KnockOut Serum Replacer (KSR, proprietary formulation; Invitrogen, Carlsbad, CA). [1][2][3] Other factors suggested to play a role in supporting the maintenance of hESCs include transforming growth factor ␤1 (TGF␤1), 4 activin A (ActA), 5,6 platelet-derived growth factor (PDGF) and sphingosine-1-phosphate, 7 BIO, a small-molecule inhibitor of GSK3␤, 8 and neurotrophins. 9 Several defined medium systems have been described for hESCs and are based upon FGF2 in combination with nodal, 10 TGF␤1, GABA, and pipecolic acid, plus lithium chloride, 11 Wnt3a plus April/BAFF, 12 or the N2/B27 supplements. 13 Although these studies have focused on identifying growth factors and conditions that support the proliferation of undifferentiated hESCs, little is known about the cell-surface receptors that are activated when hESCs are exposed to conditions favorable for self-renewal. A number of receptor tyrosine kinases (RTKs) are expressed at high levels on hESCs, 14 including insulin-like growth factor-1 receptor (IGF1R), fibroblast growth factor receptor (FGFR1), and EPHA1, as well as ERBB2 and ERBB3 (which are members of the epidermal growth factor receptor [EGFR] family), while expression of FGFR2 (EGFR) FGFR4, vascular endothelial growth factor receptor-2 (VEGFR2), IGFR2, KIT, and RET has also been detected. 15,16 RTKs are likely to be central signaling effectors 17 that influence survival, apoptosis, proliferation, or differentiation decisions in pluripotent cells. To determine if any of these RTKs are involved in self-renewal, we simultaneously interrogated the tyrosine phosphorylation status of 42 RTKs in hESCs grown in MEF-conditioned medium (CM) and developed a defined medium for hESC culture. The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 7, 2018. by guest www.bloodjournal.org From Materials and methods Cell cultureThe National Institutes of Health (NIH)-registered H1, BG01, BG02, and BG03 hESC lines, as well as CyT49, an hESC line isolated using human feeder cells under good manufacturing process (GMP) conditions (Novocell, San Diego, CA), we...

The recent development of porcine induced pluripotent stem cells (piPSCs) capable of generating chimeric animals, a feat not previously accomplished with embryonic stem cells or iPSCs in a species outside of rodents, has opened the doors for in-depth study of iPSC tumorigenicity, autologous transplantation, and other key aspects to safely move iPSC therapies to the clinic. The study of iPSC tumorigenicity is critical as previous research in the mouse showed that iPSCderived chimeras possessed large numbers of tumors, rising significant concerns about the safety of iPSC therapies. Additionally, piPSCs capable of generating germline chimeras could revolutionize the transgenic animal field by enabling complex genetic manipulations (e.g., knockout or knockin of genes) to produce biomedically important large animal models or improve livestock production. In this study, we demonstrate for the first time in a nonrodent species germline transmission of iPSCs with the live birth of a transgenic piglet that possessed genome integration of the human POU5F1 and NANOG genes. In addition, gross and histological examination of necropsied porcine chimeras at 2, 7, and 9 months showed that these animals lacked tumor formation and demonstrated normal development. Tissue samples positive for human POU5F1 DNA showed no C-MYC gene expression, further implicating C-MYC as a cause of tumorigenicity. The development of germline-competent porcine iPSCs that do not produce tumors in young chimeric animals presents an attractive and powerful translational model to study the efficacy and safety of stem cell therapies and perhaps to efficiently produce complex transgenic animals. STEM CELLS

No abstract

We used inhibitors of sphingolipid and glycosphingolipid (GSL) biosynthesis to block the generation of SSEA-3 and -4 in hESCs. Depletion of these antigens and their precursors was confirmed using immunostaining, flow cytometry, and tandem mass spectroscopy. Transcriptional analysis, immunostaining, and differentiation in vitro and in teratomas indicated that other properties of pluripotency were not noticeably affected by GSL depletion. These experiments demonstrated that the GSLs recognized as SSEA-3 and -4 do not play critical functional roles in maintaining the pluripotency of hESCs, but instead suggested roles for this class of molecules during cellular differentiation. STEM CELLS 2007;25:54 -62

Feline immunodeficiency virus (FIV) is a natural infection of domestic cats that results in acquired immunodeficiency syndrome resembling human immunodeficiency virus (HIV) infection in humans. The worldwide prevalence of FIV infection in domestic cats has been reported to range from 1 to 28%. Hence, an effective FIV vaccine will have an important impact on veterinary medicine in addition to being used as a small animal AIDS model for humans. Since the discovery of FIV reported in 1987, FIV vaccine research has pursued both molecular and conventional vaccine approaches toward the development of a commercial product. Published FIV vaccine trial results from 1998 to the present have been compiled to update the veterinary clinical and research communities on the immunologic and experimental efficacy status of these vaccines. A brief report is included on the outcome of the 10 years of collaborative work between industry and academia which led to recent USDA approval of the first animal lentivirus vaccine, the dual-subtype FIV vaccine. The immunogenicity and efficacy of the experimental prototype, dual-subtype FIV vaccine and the efficacy of the currently approved commercial, dual-subtype FIV vaccine (Fel-O-Vax FIV) are discussed. Potential cross-reactivity complications between commercial FIV diagnostic tests, Idexx Snap Combo Test and Western blot assays, and sera from previously vaccinated cats are also discussed. Finally, recommendations are made for unbiased critical testing of new FIV vaccines, the currently USDA approved vaccine, and future vaccines in development.